Abstract It is estimated that worldwide, 90% of the adult population are infected with one or more of the human herpesviruses. These viruses are responsible for lifelong debilitating and congenital infections, and some members of this family are associated with human cancers and age-related cognitive decline. Herpes simplex virus (HSV1) has infected more than 3.7 billion people under the age of 50 (67% of the population), and HSV1 and HSV2 and encephalitis) cause significant disease during acute infection (oral and genital lesions, corneal blindness establishing persistent latent infections in sensory neurons for the life of the host with the potential for reactivation and recurrent disease. Human and other vertebrate hosts have evolved a variety of strategies to sense, evade and defend themselves against viral infections, including the deployment of intrinsic antiviral restriction factors. The subject of this proposal is the intrinsic antiviral promeylocytic leukemia protein (PML) (also known as TRIM 19), an interferon-inducible RING finger SUMO ligase that has been implicated many cellular processes including protecting cells from cancer and microbial infections. During HSV infection, PML is recruited to viral genomes, triggering the formation of virally-induced nuclear bodies (viPML-NBs) that are associated with repression of viral gene expression. Surprisingly, the signal that triggers the formation of viPML-NBs has never been identified, and this represents a large gap in our knowledge of the mechanism of action of this important intrinsic restriction factor. Recent reports suggest that PML is sensitive to oxidation and forms multimers in response to oxidative stress. The formation of stress induced PML-NBs (siPML-NBs) is triggered in response to oxidation, which in turn causes the formation of covalent disulfide bonds in PML. Several enveloped viruses including HSV have been shown to increase the oxidation state of infected cells. In this proposal we will test the hypothesis that multimerization of PML and formation viPML-NBs is dependent on oxidation induced by HSV infection. Furthermore, we suggest that cysteine residues in PML are required for the formation of viPML-NBs via disulfide bond formation. In Aim 1 we will determine the mechanism of multimerization of PML by expressing individual domains alone and in combination and testing the effect of redox on their structure, solubility, integrity and multimerization state. We have constructed a 3D model of the PML homodimer and have predicted surface exposed cysteines on PML that may play a role in multimerization. These predictions will be tested by genetic and biochemical experiments. In Aim 2 we will test the hypothesis that the formation of viPML-NBs is dependent on oxidation induced by HSV infection and will test whether mutations in cysteine residues are defective in PML multimerization and viPML-NB formation.